The invention relates to the field of molecular biology and microbiology. More specifically, a 12 kb gene cluster has been isolated from Rhodococcus erythropolis HL PM-1 containing several open reading frames implicated in the degradation of picric acid.
Picric acid (2,4,6-trinitrophenol) is a compound used in a variety of industrial applications including the manufacture of explosives, aniline, color fast dyes, pharmaceuticals and in steel etching. Picric acid and ammonium picrate were first obtained as fast dyes for silk and wool. However, the unstable nature of picric acid was soon exploited for use as an explosive and explosive boosters where it is the primary component of blasting caps which are used for the detonation of 2,4,6-trinitrotoluene (TNT). Because of its explosive nature, disposal of waste picric acid poses unique hazard not generally associated with other environmental toxicants.
Mounting public concern and increasing government regulations have provided the impetus for a safe, effective means to remediate picric acid contaminated environments. Past methods of disposing of munitions and other wastes containing picric acid have included dumping at specified land-fill areas, isolation in suitable, reinforced containers, land based deep-welling, dumping in deep water at sea and incineration. All of these methods carry some potential for harm to the environment. For example, incineration creates a problem of air pollution and disposal on land risks the possibility that toxic substances will elute or leach into locations where they may threaten aquatic life forms, animals or humans. A more desirable disposal method might incorporate a chemical or enzymatic degradative process.
The metabolic reduction of organic nitrogen groups has been known for some time. Wesifall (J. Pharmacol Exp. Therap. 78:386 (1943)) reported that liver, kidney and heart tissue are active in the reduction of trinitrotoluene, however, was not able to identify the specific enzyme system responsible. Westerfield et al. (J. Biol. Chem. 227:379 (1957)) further disclosed that purified xanthine oxidase is capable of reducing organic nitrogen groups and demonstrated that the molybdenum (Mo) co-factor was essential in the degradative process.
Microbial degradation of organic nitrogen compounds has been limited to a handful of organisms. Erickson (J. Bacteriol. 41:277 (1941)) reported that certain strains of Micromonospora were able to utilize picric acid and trinitro-resorcinol as a carbon source and Moore (J Gen. Microbiol., 3:143 (1949)) described two unspecified Proactinomnycetes as being capable of using nitrobenzene as a simultaneous source of carbon and nitrogen. Gundersden et al. (Acta. Agric. Scand. 6:100 (1956)) described the metabolism of picric acid by Corynebacterium simplex which was isolated from soil as a 4,6-dinitro-2-methylphenol-degrading organism. Degradation was determined by measuring the amount of nitrate produced when the organism was contacted with an organic nitrogen compound. The extent of degradation and the identification of specific degradation products were not reported. Later, Wyman et al. (Appl. Environ. Microbiol. 37(2):222 (1979)) found that a strain of Pseudomonas aeruginosa reduced picric acid to 2-amino-4,6-dinitrophenol (picramic acid) under anaerobic conditions. Wyman further determined that degradation products from both picric and picramic acid produced by this strain demonstrated mutagenicity as assayed by the standard AMES test.
Another Pseudomonas sp., Pseudomonas putida, has been shown to be able to use picric acid as a carbon source and achieve some bio-conversion of the compound to 1,3,5-trinitrobenzene, 2,4,6-trinitroaldehyde, and 3,5-dinitrophenol (Kearney et al., Chemosphere, 12 (11-12):1583 (1983)).
Recently, Rhodococcus erythropolis has been identified a picric acid degrading bacteria. Lenke et al. (Appl. Environ. Microbiol. 58(9):2933 (1992)) teach that Rhodococcus erythropolis, under aerobic conditions, can incompletely utilize picric acid as a nitrogen source producing nitrite and 2,4,6-trinitrocyclohexanone, which cannot be degraded further. More recently a consortium of bacteria comprising members of the genera Arthrobacter, Avrobacterium and Pseudomonas has been described that has the ability to completely degrade picric acid (U.S. Pat. No. 5,543,324). Similarly, U.S. Pat. No. 5,478,743 teaches Arthrobacter isolates having the ability to mineralize picric acid and other tri-nitrophenol compounds. In work growing out of these discoveries Ebert et al. (J. Bacteriol. 181(9):2669-2674 (1999)) describe some of the possible intermediates in the picric acid bio-degradation pathway and teach the N-terminal sequence of an NADPH-dependent F420 reductase. No nucleotide sequence is disclosed and no description of other elements of the pathway are provided.
Although several wild type organisms having some ability to degrade picric acid and other nitroaromatics, have been described, to date, no genes have been identified or isolated from these or other organisms that might comprise a bio-degradative pathway for this persistent pollutant The ability to manipulate the genes involved in the picric acid degradation pathway will greatly advance the art of picric acid remediation. If such genes are known, they may be transformed into suitable hosts and overexpressed in a manner so as to optimize the degradative process.
The problem to be solved therefore is to isolate genes involved in picric acid degradation for their eventual use in creating transformants with enhanced ability to degrade picric acid. Applicants have solved the stated problem by isolating a 12 kb DNA fragment containing ten open reading frames (ORF) which have distinct homology to genes expected to play significant role in the picric acid degradative pathway.
The present invention provides isolated nucleic acid fragments encoding enzymes of the picric acid degradation pathway corresponding to ORF""s 3, 5, 6,8, 9, 10 and 11 of the present 12 kb gene cluster where the isolated nucleic acid fragments are independently selected from the group consisting of (a) isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence as set forth in SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:25; (b) isolated nucleic acid fragments that are substantially similar to isolated nucleic acid fragments encoding all or a substantial portion of the amino acid sequences as set forth in SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:25; (c) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C. and washed with 2xc3x97SSC, 0.1% SDS followed by 0.1xc3x97SSC, 0.1% SDS and; (d) and isolated nucleic acid fragments that are complementary to (a), (b) or (c).
The invention further provides the nucleic acid fragment embodying the 12 kb gene cluster comprising ORF""s 1-12 of the instant invention, useful for the degradation of picric acid.
The invention also provides chimeric genes comprised of the instant nucleic acid fragments and suitable regulatory sequences as well as the polypeptides encoded by said sequences.
The invention further provides methods for obtaining all or a portion of the instant sequences by either primer directed amplification protocols or by hybridization techniques using primers or probes derived from the instant sequences.
Additionally the invention provides recombinant organisms transformed with the chimeric genes of the instant invention and methods of the degrading picric acid and dinitrophenol using said recombinant organisms.
The invention further provides a method for the conversion of picric acid to dinitrophenol comprising: contacting a transformed host cell under suitable growth conditions with an effective amount of picric acid whereby dinitrophenol is produced, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:21 under the control of suitable regulatory sequences.
In another embodiment the invention provides a mutated bacterial gene encoding an F420/NADPH oxidoreductase or an F420-dependent picric/2,4-DNP reductase, having an altered F420 dependent reductase activity produced by a method comprising the steps of (i) digesting a mixture of nucleotide sequences with restriction endonucleases wherein said mixture comprises:
a) a bacterial gene encoding a F420/NADPH oxidoreductase or an F420-dependent picric/2,4-DNP reductase;
b) a first population of nucleotide fragments which will hybridize to said wildtype bacterial sequence;
c) a second population of nucleotide fragments which will not hybridize to said wildtype bacterial sequence;
wherein a mixture of restriction fragments are produced; (ii) denaturing said mixture of restriction fragments; (iii) incubating the denatured said mixture of restriction fragments of step (ii) with a polymerase; and (iv) repeating steps (ii) and (iii) wherein a mutated bacterial gene is produced encoding a protein having an altered F420 dependent reductase activity.